The long-term goal of the proposed research is to understand how transcription factors control cell fate and cell death in higher animals. In the last ten years, a number of transcription factors have been found that play an important role in both development and oncogenesis. Many of these genes are highly conserved in evolution, and control a variety of different developmental events in both vertebrates and invertebrates. The GLI3 gene is a Zn-finger transcription factor that was originally isolated because of its close homology to an oncogene amplified in human gliomas (GLI). Highly related genes are present in Drosophila and C. elegans, and play a key role in patterning or set determination in these different organisms. In mammals, mutations in the GLI3 gene alter patterns of programmed cell death and lead to overgrowth of both neural and limb tissue. The normal targets of GLI-like genes are largely unknown, although candidate genes have been identified in both Drosophila and mice. Here we propose to carry out a direct search for target genes that mediate GLI3 functions during limb development. We will focus on the developing digits for two reasons. First, an association between GLI3 and programmed cell death is particularly clear in this region. GLI3 is expressed in the regions where cells are known to die, and defects in GLI3 alter the patterns of cell death both between and inside the digits. Second, a candidate gene has already been identified whose expression pattern and mutant phenotype suggest that it may be a target of GLI3. The candidate gene, growth/differentiation factor 5 (GDF5), encodes a secreted signalling molecule that is structurally related to suspected targets of GLI-like genes in Drosophila. GDF5 is also expressed in digit regions where cells undergo programmed cell death, and mutations in GDF5 produce phenotypes that resemble those of GLI3 in the digit region. Here we propose to examine the possible regulatory relationships between these genes using null mutations available in mice, and an organ culture system that recapitulates many of the developmental events in the digit region. Potential target genes for GLI3 and GDF5 will be isolated by screening for genes that are differentially expressed in limb buds of GLI3 and GDF5 mutants, or in organ cultures directly exposed to the GDF5 gene product. A yeast screen will be carried out to identify genes that contain direct GLI3 response elements. Candidate target genes will be sequenced, surveyed for their expression in digits and other tissues, and tested for their effects on programmed cell death in cultured cell lines. The results of the study should provide a new understanding of the molecular events that control cell fate and cell death decisions in response to GLI3. The nature of the target genes may suggest new tests or treatments for human cancers that are caused by abnormal expression of master regulatory transcription factors.
